Antibacterial activity of lysozyme-chitosan oligosaccharide conjugates (LYZOX) against Pseudomonas aeruginosa, Acinetobacter baumannii and Methicillin-resistant Staphylococcus aureus

Abstract

The recent emergence of antibiotic-resistant bacteria requires the development of new antibiotics or new agents capable of enhancing antibiotic activity. This study evaluated the antibacterial activity of lysozyme-chitosan oligosaccharide conjugates (LYZOX) against Pseudomonas aeruginosa, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA), which should resolve the problem of antibiotic-resistant bacteria. Bactericidal tests showed that LYZOX killed 50% more P. aeruginosa (NBRC 13275), A. baumannii and MRSA than the control treatment after 60 min. In addition, LYZOX was shown to inhibit the growth of P. aeruginosa (NBRC 13275 and PAO1), A. baumannii and MRSA better than its components. To elucidate the antibacterial mechanism of LYZOX, we performed cell membrane integrity assays, N-phenyl-1-naphthylamine assays, 2-nitrophenyl β-D-galactopyranoside assays and confocal laser scanning microscopy. These results showed that LYZOX affected bacterial cell walls and increased the permeability of the outer membrane and the plasma membrane. Furthermore, each type of bacteria treated with LYZOX was observed by electron microscopy. Electron micrographs revealed that these bacteria had the morphological features of both lysozyme-treated and chitosan oligosaccharide-treated bacteria and that LYZOX destroyed bacterial cell walls, which caused the release of intracellular contents from cells. An acquired drug resistance test revealed that these bacteria were not able to acquire resistance to LYZOX. The hemolytic toxicity test demonstrated the low hemolytic activity of LYZOX. In conclusion, LYZOX exhibited antibacterial activity and low drug resistance in the presence of P. aeruginosa, A. baumannii and MRSA and showed low hemolytic toxicity. LYZOX affected bacterial membranes, leading to membrane disruption and the release of intracellular contents and consequent bacterial cell death. LYZOX may serve as a novel candidate drug that could be used for the control of refractory infections.

Fig 1. Assays for bactericidal activity.

(a) Pseudomonas aeruginosa (NBRC 13275), (b) Pseudomonas aeruginosa (PAO1), (c) Acinetobacter baumannii (JCM 6841), (d) MRSA (IID 1677). Each type of bacteria was incubated with LYZOX solution (2,000 μg/mL), the mixed solution (lysozyme solution [1,000 μg/mL] and chitosan oligosaccharide [COS] solution [1,000 μg/mL]) or the lysozyme-galactomannan conjugate (LGC) solution (2,000 μg/mL) in saline at 37°C in a water bath for 0 min, 30 min, 60 min and 120 min. The dilutions were plated, and the colonies were counted following growth overnight. The values are the mean ± SEM from four independent experiments. *p<0.05 or **p<0.01 compared with saline; †p<0.05 or ††p<0.01 compared with LGC; ‡p<0.05 or ‡‡p<0.01 compared with the mixture (unpaired t-test). Symbols: circles, saline; squares, lysozyme-chitosan oligosaccharide conjugate (LYZOX); up-pointing triangles, LGC; down-pointing triangles, mixture.

Fig 2. Growth inhibition test.

(a) Pseudomonas aeruginosa (NBRC 13275), (b) Pseudomonas aeruginosa (PAO1), (c) Acinetobacter baumannii (JCM 6841), (d) MRSA (IID 1677). Each type of bacteria was incubated with LYZOX solution (2,000 μg/mL), lysozyme solution (1,000 μg/mL), chitosan oligosaccharide (COS) solution (1,000 μg/mL), the mixed solution (lysozyme solution [1,000 μg/mL] and COS solution [1,000 μg/mL]) or lysozyme-galactomannan conjugate solution (2,000 μg/mL) in tryptic soy broth (TSB) at 37 °C for 6 h. The dilutions were plated, the colonies were counted following growth overnight, and results were compared with the control. The control was TSB without each treatment. The values are the mean ± SEM from 18 independent experiments for P. aeruginosa (NBRC 13275). The values are the mean ± SEM from eight independent experiments for P. aeruginosa (PAO1), A. baumannii and MRSA. *p<0.05, **p<0.01 (unpaired t-test). LYZOX: lysozyme-chitosan oligosaccharide conjugate. LGC: lysozyme-galactomannan conjugate.

Fig 3. Cell membrane integrity assays.

The measurement of the absorbance at 260 nm (A_{260 nm}) can be used to estimate the amount of nucleic acids released from the cytoplasm, which can be used to evaluate the cell membrane integrity. (a) Pseudomonas aeruginosa (NBRC 13275), (b) Acinetobacter baumannii (JCM 6841), (c) MRSA (IID 1677). The values are the mean ± SEM of triplicate measurements. *p<0.05, **p<0.01 (unpaired t-test).

Fig 4. NPN assays.

The outer membrane permeability of the gram-negative bacteria and plasma membrane permeability of MRSA were determined by NPN assays. (a) Pseudomonas aeruginosa (NBRC 13275), (b) Acinetobacter baumannii (JCM 6841), (c) MRSA (IID 1677). The values are the mean ± SEM. Three independent experiments were performed for each type of bacteria in the control solution and at each concentration of LYZOX. *p<0.05, **p<0.01 (unpaired t-test). RFU: relative fluorescence units.

Fig 5. ONPG assays.

The measurement of absorbance at 420 nm (A_{420 nm}) can be used to estimate the activity of cytoplasmic β-galactosidase released from bacteria into LYZOX solution using ONPG as the substrate to evaluate inner membrane permeabilization in gram-negative bacteria and plasma membrane permeabilization in gram-positive bacteria. (a) Pseudomonas aeruginosa (NBRC 13275), (b) Acinetobacter baumannii (JCM 6841), (c) MRSA (IID 1677). The values are the mean ± SEM of triplicate measurements. *p<0.05, **p<0.01 (unpaired t-test).

Fig 6. LIVE/DEAD bacterial viability assays.

(a) Pseudomonas aeruginosa (NBRC 13275), (b) Acinetobacter baumannii (JCM 6841), (c) MRSA (IID 1677). Each type of bacteria was incubated in lysozyme-chitosan oligosaccharide conjugate (LYZOX) solution (1,000 μg/mL) or saline at 37°C for 2 h. Bacteria with intact cell membranes were stained with green fluorescent SYTO 9, whereas bacteria with damaged membranes were stained with red fluorescent propidium iodide (PI). The excitation/emission wavelengths were 488/495-515 nm for SYTO 9 and 488/635-700 nm for PI. Scale bar = 25 μm.

Fig 7. Scanning electron microscopic findings in Pseudomonas aeruginosa, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus.

Scanning electron micrographs of Pseudomonas aeruginosa (NBRC 13275) (a, b, c, d, e), Acinetobacter baumannii (JCM 6841) (f, g, h, i, j) and MRSA (IID 1677) (k, l, m, n, o) incubated with each treatment for 2 h. (a), (f) and (k) were not treated (control). (b), (g) and (l) were treated with lysozyme-chitosan oligosaccharide conjugate (LYZOX, 1,000 μg/mL). (c), (h) and (m) were treated with lysozyme (500 μg/mL). (d), (i) and (n) were treated with chitosan oligosaccharide (COS, 500 μg/mL). (e), (j) and (o) were treated with the mixture (lysozyme [500 μg/mL] and COS [500 μg/mL]). Bacteria were photographed at a magnification of ×20,000. Scale bar = 1.5 μm.

Fig 8. Transmission electron microscopic findings in Pseudomonas aeruginosa, Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus.

Transmission electron micrographs of Pseudomonas aeruginosa (NBRC 13275) (a, b, c, d, e), Acinetobacter baumannii (JCM 6841) (f, g, h, i, j) and MRSA (IID 1677) (k, l, m, n, o) incubated with each treatment for 2 h. (a), (f) and (k) were not treated (control). (b), (g) and (l) were treated with lysozyme-chitosan oligosaccharide conjugate (LYZOX, 1,000 μg/mL). (c), (h) and (m) were treated with lysozyme (500 μg/mL). (d), (i) and (n) were treated with chitosan oligosaccharide (COS, 500 μg/mL). (e), (j) and (o) were treated with the mixture (lysozyme [500 μg/mL] and COS [500 μg/mL]). Bacteria were photographed at a magnification of of ×50,000. Scale bar = 0.2 μm.

Fig 9. Magnesium effect.

(a) Pseudomonas aeruginosa (NBRC 13275), (b) MRSA (IID 1677). Each type of bacteria was incubated with each treatment solution (LYZOX [10,000 μg/mL] and COS [10,000 μg/mL]) and magnesium (0.05 M) in tryptic soy broth (TSB) at 37 °C for 10 h. Then, each solution was measured spectrometrically at 600 nm (A_{600 nm}) and compared with the value of the control. The control was TSB without each treatment. In order to evaluate the effect of magnesium, we performed the same experiments without magnesium. The values are the mean ± SEM from three independent experiments for P. aeruginosa (NBRC 13275) and MRSA. *p<0.05, **p<0.01 (unpaired t-test). LYZOX: lysozyme-chitosan oligosaccharide conjugate. COS: chitosan oligosaccharide. Mg: magnesium.

Fig 10. Acquired drug resistance test.

Bacteria were subcultured repeatedly in LB broth containing lysozyme-chitosan oligosaccharide conjugate (LYZOX) or the mixture (lysozyme and chitosan oligosaccharide). They were evaluated for their change of susceptibility to LYZOX or mixture. The control was not subcultured. (a) Pseudomonas aeruginosa (NBRC 13275), (b) Pseudomonas aeruginosa (PAO1), (c) Acinetobacter baumannii (JCM 6841), (d) MRSA (IID 1677). For MRSA, the test of mixture was finished because antibacterial resistance of the mixture was confirmed at the fourth transfer. Symbols: filled circles, LYZOX, subcultured; filled rhombuses, LYZOX, control; open circles, mixture, subcultured; open rhombuses, mixture, control.

Fig 11. Heat stability of lysozyme and LYZOX.

(a) The effects of heat were tested with lysozyme-chitosan oligosaccharide (LYZOX) and lysozyme exposed to 80°C for various times. The lytic activity of heated agents against Micrococcus luteus for 10 min were measured at OD₆₀₀. The values are the per cent of residual activity. Symbols: filled circles, LYZOX, filled squares, lysozyme. (b) Comparison of lytic activity of LYZOX and lysozyme for 120 min at 80°C. Lytic activity against Micrococcus luteus was measured at OD₆₀₀. Symbols: open circles, non-heated LYZOX; filled circles, heated LYZOX; open squares, non-heated lysozyme; filled squares, heated lysozyme.